PROJECT SUMMARY Selective interactions between neurons and non-neuronal cells are crucial for the development and function of neural circuits. Pain-sensing somatosensory neurons project peripheral axons to the skin, where they branch extensively amongst epithelial epidermal cells. Although these sensory terminals are called ?free endings?, recent studies have revealed that they are often wrapped by epidermal cells, which enclose them into ensheathment channels reminiscent of those formed by non-myelinating Remak Schwann cells. Although underappreciated, epidermal ensheathment channels have been observed in worms, flies, fish, and mammals, indicating that ensheathment is a conserved feature of epidermal sensory endings, and thus likely plays critical roles in the development and function of nociceptive axons. Little is known about the morphogenetic process of axon ensheathment by epidermal cells, and nothing is known about how these structures contribute to sensory function or disease in vertebrate animals. This proposal investigates the morphogenetic mechanisms that create epidermal ensheathment channels and how they contribute to sensory function in zebrafish larvae. Their external development and the availability of unique transgenic tools make zebrafish an ideal model for studying this dynamic morphogenetic process. Preliminary work using live fluorescent reporters for subcellular structures in zebrafish skin cells identified a sequence of events leading up to ensheathment and suggested a step-wise morphogenetic process. First, axons growing into this epidermis induce the formation of specialized lipid microdomains at skin cell-axon contact sites. Second, F-actin is recruited to these microdomains, likely promoting membrane invagination to initiate the ensheathment process. Finally, adherens junctions and desmosomes form at ?necks? of ensheathment channels to tightly seal the channels. The first two aims of this proposal use innovative microscopy approaches for high spatial and temporal resolution live imaging, cell-specific molecular manipulations, and CRISPR/Cas9-mediated mutagenesis to determine how axons and skin cells establish selective interactions and execute the ensheathment process. These studies will illuminate morphogenetic mechanisms relevant not just to axon ensheathment by epidermal and glial cells, but also to basic cellular processes, like the formation of membrane signaling domains and junction assembly. The third aim combines imaging and behavioral assays to reveal how axon ensheathment impacts neuronal structure and function. These studies have the potential to uncover a critical feature of the touch-sensing apparatus and suggest how ensheathment contributes to disease conditions affecting pain and touch sensation.